Potential of hydroethanolic leaf extract of Ocimum sanctum in ameliorating redox status and lung injury in COPD: an in vivo and in silico study

Oxidative stress and inflammation are hypothesised as the main contributor for Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke (CS), a major cause of COPD leads to inflammation resulting in recruitment of neutrophils and macrophages which are rich sources of oxidants. Activation of these cells produces excess oxidants and depletes antioxidants resulting in stress. Presently, effective drug for COPD is limited; therefore, novel compounds from natural sources, including plants are under exploration. The present study aims to investigate the protective effect of Ocimum sanctum leaf extract (OLE) in CS − induced model of COPD. Exposure to CS was performed thrice a week for 8 weeks and OLE (200 mg/kg and 400 mg/kg) was administered an hour before CS exposure. Control group (negative control) were exposed to ambient air while COPD group was exposed to CS (positive control). Administration of OLE doses reduced inflammation, decreased oxidant concentration and increased antioxidant concentration (p < 0.01). Molecular docking studies between the major phytocompounds of OLE (Eugenol, Cyclohexane and Caryophyllene) and antioxidant enzymes Superoxide dismutase (SOD), Catalase, Glutathione peroxidase (GPx), Glutathione reductase (GR) and Glutathione S Transferase (GST) showed strong binding interaction in terms of binding energy. In vivo and in silico findings for the first time indicates that OLE extract significantly alleviates oxidative stress by its potent free radical scavenging property and strong interaction with antioxidant enzymes. OLE extract may prove to be a therapeutic option for COPD prevention and treatment.


Results
OLE inhibited the airway inflammation by suppressing total and differential cell count. Total cells were counted in BALF to evaluate cellular infiltration into lungs. Remarkable increase in the number of total leukocyte recruitments was observed in COPD experimental mice as compared to the normal mice (Fig. 1A). The recruited cells mainly include the neutrophils (43%) and macrophages (32%) as counted on the cytospin slides (Fig. 1B,C). Pre − treatment with OLE downregulated the inflammation in the lungs (p < 0.05). This is supported by significant decrease in the accumulation of inflammatory cells accompanied with reduce neutrophils and macrophages. TOS and TAS regulation by OLE. TOS was increased in COPD mice as compared to the normal mice whereas downregulation was found with OLE. There was an increase of 36.6% of total oxidants in the COPD mice as compared to normal mice (p < 0.05). The administration of OLE at 200 mg/kg and 400 mg/kg reduced the level of TOS to 8% and 59% respectively. TAS was declined in COPD mice to 42.5% while OLE at 200 mg/kg and 400 mg/kg upregulated the antioxidant status to 48% and 52.7% respectively (p < 0.01) ( Fig. 2A,B). www.nature.com/scientificreports/ Protein and protein carbonyl content in BALF and lung. The total protein and protein carbonyl concentration was found to be significantly elevated in BALF as well as lung homogenate of CS group as compared to the control group (p < 0.05). OLE treatment significantly attenuated the CS − induced enhanced total protein and protein carbonyl content in BALF as well as lung homogenate (p < 0.01 and p < 0.05) (Fig. 3A,B).  Results were analysed statistically by one way ANNOVA followed by Turkey's test. *p < 0.01 and **p < 0.05 versus the normal group; # p < 0.01 and ## p < 0.05 versus the COPD group.

Modulation in lung antioxidant activity (SOD, Catalase, GPx, GR, GSH and GST). As depicted
in Fig. 5A-C, the CS − induced COPD group exhibited significantly reduced activity of SOD (p < 0.05), Catalase (p < 0.05) and GPx (p < 0.01) as compared to the control group. However, administration of OLE significantly restored the activity of SOD (p < 0.05), Catalase (p < 0.05) and GPx to normal (p < 0.01). In contrast, the GR activity (p < 0.05) and GSH level (p < 0.01) were substantially elevated by CS exposure (p < 0.01). OLE treatment reduced GR activity and GSH level (Fig. 5D,E). OLE 200 mg/kg did not have any effect on GR activity. GST activity was also downregulated in CS exposed mice whereas upregulation in the activity was found with OLE treatment (Fig. 5F). OLE 400 mg/kg more effectively modulated the GST activity compared to 200 mg/kg. The antioxidative effect of OLE at 400 mg/kg was better than that of OLE at 200 mg/kg and standard drug.
OLE inhibited MDA level. CS − induced COPD model showed adaptive increase in MDA level in the lungs as compared with the control group (p < 0.05). However, treatment with OLE (200 and 400 mg/kg) significantly decreased the levels of MDA content (p < 0.01). Moreover, treatment with prednisolone also significantly suppressed the elevated MDA content in lung homogenate (Fig. 6).
Emphysema and alveolar destruction. H&E staining was performed to evaluate the morphometric pathological changes in the lung tissue in terms of inflammation, emphysema and DI. As shown in Fig. 8A,B, the lung tissue mainly the alveolar spaces was significantly damaged as marked by enlarged airspaces and peribronchiolar inflammation was observed as a consequence of CS exposure. OLE and prednisolone modulated the alveolar destruction and peribronchiolar inflammation. L m and % DI representing emphysematous changes, quantified in histological sections were significantly increased in CS − exposed mice compared to the control mice (p < 0.05 and p < 0.01) (Fig. 8C,D). However, lungs of OLE 400 mg/kg treated mice showed significantly lower L m and % DI values compared to CS − exposed mice (p < 0.05).

Discussion
In the present investigation, the protective effect of OLE was studied in COPD induced by CS. For this, in vivo experiments were performed to demonsrate the downregulaion of inflammation and oxidative sress by OLE in murine model. The finding of in vivo experimens were further supported by in silico validation. CS has been used as an inducer to provoke the pathophysiology of COPD in mouse model and study the alterations in the airways as an imbalance of oxidant generation and antioxidant resistance and inflammation. Our finding provide evidences that OLE can be useful to suppress various pathological features associated with COPD. To the best of our knowledge this is the first study of its kind to report the potency of OLE in attenuating the inflammation and oxidative damage in CS − induced COPD mice by using both invivo and in silico approaches. Prednisolone, www.nature.com/scientificreports/ widely used as an anti − inflammatory drug, has been used as a positive control in investigating pulmonary dysfuntioning as a consequence of inflammation and oxiadative stress in COPD.
Numerous studies reported high content of toxic substance, potent oxidants and free radicals including quinone, hydroquinone, aldehyde, semi quinone and superoxide in cigeratte smoke 19 . Entrance of any irritant including CS into the respiratory tract alters the alveolar enviornment leading to epithelial cell damage and production of several cytokines and chemokines 20 . These cytokines and chemokines are responsible for inflammatory cell infiltration including neutrophils and macrophages and further their activation, leading to severe inflammatory response in airways 20 . The present investigation reports significant increase in infiltration and recruitment of immune cells (neutrophils and macrophages) in CS exposed mice where OLE treatment attenuated the inflammation by inhibiting the recruitment of cells including neutrophils and macrophages. , GSH (E) activity and GST content (F) in CS exposed mice: The modulated activity of SOD, catalase, GPx, GR and GST in CS − induced mice was reverted with the administration of OLE with 200 mg/kg and 400 mg/kg. The increased content of GSH in CS − induced mice was effectively decreased with the administration of OLE 200 mg/kg and 400 mg/kg. Values are expressed as mean ± SEM. Results were analysed statistically by one way ANNOVA followed by Turkey's test. *p < 0.01 and **p < 0.05 versus the normal group; # p < 0.01 and # p < 0.05 versus the COPD group.  21 . CS exposure is believed to drive ROS generation which distrupts macromolecules − DNA, protein and lipid thereby dysfunctioning many biochemical and physiological processes. Previous investigations proved ROS generation as a major marker for oxidant production trigerring inflammatory response by CS exposure. The protective effect of OLE was observed in ROS generation where enhanced production of ROS in CS − induced COPD was suppressed by OLE.
The levels of TAS and TOS are considered as marker of oxidative stress 22 . Most interestingly the present study also showed an upregulation in TOS and downregulation in Trolex equivalent antioxidant status (TEAS) in COPD mice which signifies imbalance of oxidants and antioxidants. The present result is also consistent with many of the previous studies where enhanced TOS and suppressed TAS in COPD subjects led to oxidants and antioxidants imbalance provoking to generate stress. OLE tends to regulate the TOS and TEAS levels suggesting its capability to maintain the balance of oxidant and antioxidant mechanism.
The prominent role of oxidative stress as a consequence of excess oxidants and reduced antioxidants defence in COPD pathophysiology is evident from several studies [18][19][20] . The generation of free radicals thereby depleting antioxidants generates an imbalance between oxidant production and antioxidant defense further initiating the phenomena of inflammation and oxidative stress 5,6,23 . It is well recognized that the endogenous antioxidant enzymatic profile constiting SOD, Catalase, GPx, GR and GST play an important role in free radical and peroxide metabolism protecting the cells against oxidant stress 22,[24][25][26][27] . SOD and catalase, two crucial antioxidant enzyme functions simultaneously as oxide radical scavenger, thereby maintaining a balance between the production and scavenging of ROS. SOD functions to detoxifies the superoxide anion radicals by converting it into H 2 O 2 and O 2 , while catalse decomposes H 2 O 2 further to water and oxygen 28,29 . GPx and GR play a critical role in GSH productionwhich acts as a reducing substrate in the redox cycle facililating the reduction of H 2 O 2 to H 2 O and O 2 by GPx [30][31][32] . Further, GST functions to inactivate reactive electrophiles in coordination with GSH dependent mechanism and GR recycles the oxidized GSSG using NADPH as the reducing co − factor, thereby maintains appropriate intracellular GSH level in the cell 32 . In the present study, the COPD mice expressed decreased activity of SOD, CAT and GPx in lung tissues which may be responsible for increased oxidative stress. The decreased Figure 6. Effect of OLE on MDA level in CS exposed mice: OLE decreased the level of MDA in lung tissue in CS − induced mice where 400 mg/kg was significantly more effective. Values are expressed as mean ± SEM. Results were analysed statistically by one − way ANNOVA followed by Turkey's test. **p < 0.05 versus the normal group; # p < 0.01 and ## p < 0.05 versus the COPD group. www.nature.com/scientificreports/ activity of SOD in COPD may be correlated with increased inflammation and ROS generation.OLE treatment modulated the antioxidant status in the tissue as evident from increased SOD, catalase and GPx. GR helps in generating GSH, hence the level of both GR and GSH was excerbated in COPD mice and suppressed in OLE treated mice.
On the other hand, GSH has also been reported to excerbate the level of NO and NO has been linked with increased neutrophilic inflammation 33 . In the present investigation, an increase in NO content was observed in COPD mice which was reduced in OLE treated group. The increased NO content might be due to increased neutrophilic inflammation and GSH level. Morever, GSH forms also conjugates with different electrophilic compounds, when the electrophile is very reactive, or more often through the action of GST. GST activity was also enhanced in COPD mice but declined with OLE treatment.
Peroxidase enzyme as MPO, released from activated neutrophil and macrophages is also considered as a potent source of ROS which catalyze the generation of hypohalous acids responsible for detrimental effect on the lungs amplifying inflammatory response 34,35 . The present study showed a sharp increase in MPO activity which was consistent with several prior investigations 36 . MPO activity in the lungs and BALF revealed inhibition by OLE treatment which might be correlated with the inhibition in ROS level also. . Control group showed no abnormalities and no destruction in the alveolar spaces and no peribronchiolar inflammation. COPD sections showed enlargement of the alveolar space with peribronchiolar inflammation. Prednisolone 1 mg/kg and OLE 200 mg/ kg represent minor improvement in the airway enlargement and peribronchiolar inflammation while OLE 400 mg/kg effectively altered the alveolar spaces with no destruction and inflammation. The Lm (C) and DI (D) also exhibited improvement by OLE treatment in CS exposed mice. Values are expressed as mean ± SEM. Results were analysed statistically by one way ANNOVA followed by Turkey's test. *p < 0.01 and **p < 0.05 versus the normal group; # p < 0.01 and ## p < 0.05 versus the COPD group. Double headed arrow ( ↔) shows alveolar enlargement; single headed arrow ( →) shows peribronchiolar inflammation. www.nature.com/scientificreports/ MDA, is considered a reliable marker of oxidative stress and has been observed as an index for lipid peroxidation further revealing the oxidative injury of cell membrane 37 . Studies indicates that increase in ROS level and decresed activity of SOD and catalase correlates with cellular membrane damage by inducing lipid peroxidation  www.nature.com/scientificreports/  www.nature.com/scientificreports/ and the observation in the present study is similar to the previous findings 38 . Also, MDA content has been positively correlated with protein carbonyl content 39 . The present study showed an increase in MDA and protein carbonyl content in COPD mice which were impeded by OLE treatment. Consistent with Th1 responses, TNF − α and IFN − γ representing synergistic effect have been reported to enhance COPD by elevating inflammation 40 . Both cytokines secreted by macrophages and T cells have been linked with the development of emphysema in mice 41 . The enhanced release of IFN − γ in COPD mice in the present study might be correlated with the increased release of TNF − α which may be further linked with the increased macrophages. OLE downregulated both cytokines, further supporting its role in regulating Th1 pathway at the local level.
Our study also demonstrates that OLE therapy significantly improved the histopathological changes in the lung tissue. As a form of lung tissue injury, alveoli with damaged parenchymal wall and peribronchiolar inflammaion appeared in CS − induced COPD mice in H&E stained section which was ameolirated after OLE treatment. Hence, OLE regulated many crucial factors of inflammation and oxidative stress thereby regulating pathological characters of COPD.
Computational tools along with experimental strategies have been of great value in modern drug design and development of novel promising compounds. Using computational tool model, we investigated possible inhibitory potential of major phytochemicalsof OLE (eugenol, cyclohexane and caryophyllene) on antioxidant defense system and compared its potentialiy with a sandard anti − inflammatory drug, prednisolone. The molecular docking, binding affinity with negative ΔG values and docking scores of eugenol, cyclohexane and caryophyllene in OLEwere comparable to prednisolone and revealed the capability of protein − targets (SOD, CAT, GPx, GR and GST: five key enzymes) binding of the antioxidant defense system. The docking results correlatesand supportes the in vivo antioxidants assay representing perfect interaction of protein − target.The present docking findings was found comparable wih initial findings obtained by Alminderej et al. where eugenol and caryophyllene (phytocompounds of extract of Piper cubeba L.) exhibited potential inhibitory effecton human protein target peroxiredoxin5 42 . Thus, it may besumarized that antioxidative tendency of OLE is due to the presence of different phytoconstituents which may be responsible for its potency.
The study also has certain limitations. The present study targets the effect of OLE on inflammatory and oxidative parameters. For the reason that remodeling is one of the characteristic feature of chronic COPD, further investigations needs to be performed on the remodeling parameters. Further to evaluate the mode of action of OLE, molecular signaling mechansism needs to be studied which may be supportive for the ongoing study.

Conclusion
The present study supports the initial findings that airway inflammation and oxidative stress are connecting pathology in COPD. Our findings reflects the protective effect of OLE on the major indicators of oxidative stress, inflammation and lung injury. OLE 200 and 400 mg/kg body weight changed the oxidative/antioxidative  Mice were randomly divided into five groups (8mice/group) as in Table 2. Group I was control mice and exposed to ambient air; Group II was COPD induced mice and exposed to CS as given in the protocol below; Group III was exposed to CS and treated with Prednisolone (1 mg/kg bw); Group IV was exposed to CS and treated with OLE extract (200 mg/kg bw) and Group V was exposed to CS and treated with OLE extract (400 mg/ kg bw). Both extract and standard drug were administered intraperotonially one hour before cigarette smoke exposure. Development of COPD model by smoke induction and drug administration. Mice were exposed to CS to develop COPD in a closed chamber as per the protocol of Ghorani et al. 43 . The closed chamber consists of peristaltic pump, a fan to circulate the air into the chambers, a smoke generating chamber and a whole − body CS exposure chamber or inhalation chamber serially connected by silicone tubes. The smoke − exposed animals were subjected to two nonfiltered CS (Brand − Wills Navy cut manufactured and distributed by the ITC Limited Kolkata, India). Exposure to two CS was performed thrice a week till 8 weeks as represented in Fig. 9. The prepared leaf extract of Ocimum was administered at a dose of 200 mg/kg and 400 mg/kg. OLE was administered through intraperitoneal route in a total volume of 50 µl one hour before CS exposure. Dose determination (200 mg/kg and 400 mg/kg) for the present study was adapted from the previous studies 44,45 . Prednisolone was administered at a dose of 1 mg/kg bw through intraperitoneal route one hour before CS exposure. Twenty four hours after the last exposure, mice were euthanized. Broncheo Alveolar Lavage Fluid (BALF), lungs and blood were collected for study.

Collection of sample.
Mice were sacrificed by cervical dislocation 24 h after the last smoke exposure for collection of BALF, blood and the lungs. The trachea of the mice was cannulated and BALF was collected by injecting phosphate buffer saline (PBS). Briefly, the lung lumens were washed with 1 mL of ice chilled phosphate buffer three consecutive times and a total volume of 2.5 mL BALF was collected. Nearly 70% of the injected PBS was retrieved back in every wash. Collected BALF was centrifuged, and supernatant was used to analyse Nitric www.nature.com/scientificreports/ oxide (NO), Myeloperoxidase (MPO), protein and protein carbonyl content. BALF pellet cells were used to analyse total and differential cell count. Furthermore, the lungs were inflated with 10% Neutral buffer formalin (NBF), removed aseptically and preserved in 10% NBF for studying histological lung damage in alveolar spaces. The remaining lobes were homogenized in phosphate buffer, centrifuged at 3000 rpm and 4 °C and the supernatant was used to determine total oxidant and antioxidant status and antioxidant activities of SOD, Catalase, GPx, GR, GSH and GST. Lung lobes were also used for MPO and Malondialdehyde (MDA) study.
Total and differential cell count. BALF suspension pellet was used for total cell count by trypan blue dye exclusion test. Briefly, the cells of the pellet were stained with trypan blue and counted in haemocytometer. The remaining BALF pellet cell suspension were cyto centrifuged at 800 rpm for 5 min on gelatin coated slides for differential count. The slides were air dried, fixed in methanol and stained with Geimsa stain. Immune cells were counted and enumerated on the basis of their nuclear morphology in a total number of 100 cells.     52 . Lung tissue homogenate (10%) was prepared in 50 mM phosphate buffer containing 0.5% cetyltrimethylammonium bromide (CTAB) and centrifuged at 12,000 rpm for 30 min. The supernatant along with the pellet were subjected to three times for freeze and thaw cycle and finally centrifuged at 12,000 rpm for 15 min. MPO assay was performed in 300 µl of total volume in 96 well microplate. In brief, 20 µl of the supernatant and BALF was mixed with 280 µl reaction mixture containing 0.167 mg/mL o − dianisidine dihydrochloride and 0.002% hydrogen peroxide in 50 mM phosphate buffer. The absorbance was measured at 460 nm for 20 min in a microplate reader. MPO activity was expressed as unit/mg of tissue and measured as a change in the absorbance within 20 min.
Assessment of oxidative stress in lung homogenate. SOD activity. SOD activity was measured as previously described by Das et al. 53 . 10% lung homogenate was prepared in 50 mM phosphate buffer. Reaction mixture was prepared by mixing 1.14 mL 50 mM phosphate buffer (pH 7.4), 75 μl 20 mM α − methionine, 40 μl Triton X − 100, 75 μl 100 mM hydroxylamine hydrochloride and 100 μl 50 μM EDTA. 50 μl lung homogenate prepared in phosphate buffer was mixed with the reaction mixture and incubated for 5 min at 37 °C. Further, 80 μl of 50 μM riboflavin was added and incubated for 10 min in light inside a box coated with aluminium foil. Freshly prepared Griess reagent (1 mL) containing 1:1 solution of 0.1% NED and 1% sulphanilic acid in 5% orthophosphoric acid was added to reaction mixture. Absorbance of the mixture was taken by spectrophotometer (Aquamate, Thermo Scientific, Goteborg − Swedan) at 543 nm. SOD activity was expressed in per milligram of protein.
Catalase activity. Catalase activity was measured according to the previously described method of Aebi et al. with slight modification 54 . Reaction mixture was prepared by adding 10 µl of homogenate (prepared in 50 mM phosphate buffer), 490 µl distilled water, 1100 µl 50 mM phosphate buffer and 500 µl H 2 O 2 (60 mM). Decrease in absorbance was observed for 5 min at 290 nm. Catalase was expressed in µmoles/min/mg of protein. www.nature.com/scientificreports/ Glutathione peroxidase (GPx). GPx was measured by the established protocol with slight modification 55 . Briefly, reaction mixture was prepared by adding 0.2 mL phosphate buffer (50 mM; pH 7.0), 0.1 mL sodium azide (10 mM), 0.2 mL lung homogenate (prepared in 50 mM phosphate buffer), 0.2 mL glutathione (4 mM) and 0.1 mL 25 mM H 2 O 2 . The tubes were incubated at 37 °C for 15 min, and the reaction was terminated by the adding 0.5 mL trichloroacetic acid (10%). To determine the residual glutathione, the reaction mixture was centrifuged at 1000 rpm for 10 min. After centrifugation, 1 mL supernatant was mixed with 1 mL DTNB (80 mg/mL in 1% sodium citrate). Absorbance was read at 412 nm spectrophotometrically (Aquamate, Thermo Scientific, Goteborg − Swedan). Results were expressed as μg of GSH consumed/mg protein.
Glutathione reductase (GR). GR was estimated by the established protocol with minor modification 56 . Reaction mixture was prepared by adding 750 μl 0.2 M potassium phosphate buffer having 0.2 mM EDTA, 255 μl distilled water, 300 μl 2 mM NADPH, 75 μl oxidized glutathione (20 mM) and 20 μl lung homogenate. Absorbance was measured at 340 nm for 5 min in spectrophotometer. Decrease in absorbance indicates the activity of glutathione reductase. Results were expressed as units per mg of protein.
Reduced glutathione (GSH). GSH was detected as per the previously discussed protocol 57 . 100 μl tissue homogenate, 600 μl reaction buffer containing 0.1 M sodium phosphate buffer (pH 7.0) and 1 mM EDTA were added. Further, 760 μl distilled water and 40 μl DTNB (0.04%) dissolved in 1% sodium tricitrate were added. The reaction mixture was incubated for 5 min and absorbance was read at 412 nm. Using the standard curve, GSH concentration for each unknown sample was determined and expressed as μM/mL.

Glutathione − S − transferase (GST)
. GST activity was assayed by the standardized method of Macdonald et al., with certain modification 58 . Briefly, reaction mixture was prepared by adding 1 mL phosphate buffer (500 mM; pH 6.5), 100 µl 10% lung homogenate, 1.7 mL distilled water and 100 µl CDNB (30 mM in 95% ethanol). The mixture was then incubated at 37 °C for 10 min. After incubation, 100 µl reduced glutathione (30 mM) was added to the mixture. The mixture was centrifuged, and 1 mL of supernatant was mixed with 3 mL of reaction mixture (1.7 mL of phosphate buffer, 0.1 mL of CDNB and 1.2 mL of GSH). Absorbance was measured at 340 nm. Results were expressed as µmoles of CDNB conjugate/min/mg of protein.
Estimation of malondialdehyde (MDA). MDA is the end product of major chain reactions leading to oxidation of fatty acids, and measurement of MDA is widely used for assessing lipid peroxidation. Lipid peroxidation was studied in the lungs by measuring MDA level in the form of thiobarbituric acid active substances with slight modifications 59 . In brief, 10% lung homogenate was prepared in potassium phosphate buffer (pH 7.4) and reaction mixture was prepared by adding 50 μl of homogenate, 50 μl of 8.1% Sodium dodecyl − sulphate (SDS), 375 μl of 20% acetic acid, 375 μl of 8.1% thiobarbituric acid and 150 μl distilled water. The reaction mixture was boiled for 1 h and cooled at room temperature to develop pink color, followed by addition of 250 μl distilled water and 1.25 mL butanol and pyridine (15:1). The reaction mixture was centrifuged at 2000 rpm for 10 min, separating into two layers. The absorbance of upper layer was taken at 532 nm, and MDA concentration was expressed in nanomoles per milligram of protein.
Estimation of TNF − α and IFN − γ cytokine level. TNF − α and IFN − ƴ were measured in BALF supernatants with commercially available ELISA Kit (Biolegend, San Diego USA) as per the manufacturer's instruction. Briefly, plates were coated with capture antibody and kept overnight at 4 °C. Plates were washed and primarily incubated for blocking for 1 h, then incubated with standards and samples for 2 h, further with detection antibody for 1 h, avidin − HRP for 30 min and finally incubated with TMB substrate solution in dark for 20 − 30 min till blue color appears. The reaction was stopped by adding 1 M H 2 SO 4 . The absorbance was taken at 570 nm and subtracted with absorbance at 450 nm. At every step plates were washed 3 − 4 times with Tris buffer saline tween 20 (TBST) and incubation was performed at room temperature. Using the standard curve, concentration for each sample was determined. The results are shown in pg/mL. Absorbance was read at 450 nm. Using the standard curve, concentration for each sample was determined in pg/mL (Supplementary Table).
Lung emphysema and alveolar destruction by histological evaluation. For lung histology, the lung lobes were aseptically removed, fixed in 10% NBF, embedded in paraffin wax and 5 μm thin sections were sliced. Sections stained with haematoxylin and eosin (H&E) were used for light microscopy examination. Imaging was performed using an Olympus CX43 microscope (Tokyo, Japan). Sections with bronchioles were selected for peribronchiolar inflammation. Five full sections with 10 randomly selected fields per section were observed for evaluating emphysema (Mean Linear intercept; Lm) and destructive index (DI). Emphysema was observed as a measure of destruction of alveolar walls accompanied by damage lung parenchyma leading to enlarged alveolar spaces 60 . Quantification of air space was performed only in the sections without any cutting artifact. A grid with 40 points that were at the centre of hairline crosses was superimposed on the lung field. Structures lying under these points were classified as normal (N) or destroyed (D) alveolar spaces. DI was calculated as percentage of destroyed alveoli of all the alveoli counted per section 61 .